Abstract
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00412-012-0370-0) contains supplementary material, which is available to authorized users.
Highlights
The DNA-dependent SNF2/SWI ATPase helicase Plk1interacting checkpoint helicase (PICH) was originally identified as a binding partner and substrate of polo-like kinase 1 (Plk1), a major regulator of M phase progression (Baumann et al 2007)
Using a combination of antibody microinjection and siRNA-rescue experiments, we have explored the function of PICH during M phase progression as well as the mechanisms that contribute to control its localization at centromeres/KTs and chromosome arms, respectively
In response to both antibody microinjection and siRNA-mediated PICH depletion, we observed a substantial increase in the frequency of chromatin bridges and micronuclei formation
Summary
The DNA-dependent SNF2/SWI ATPase helicase Plk1interacting checkpoint helicase (PICH) was originally identified as a binding partner and substrate of polo-like kinase 1 (Plk1), a major regulator of M phase progression (Baumann et al 2007). PICH has become the marker of choice for monitoring threads that are commonly referred to as ultrafine DNA bridges (UFBs; Chan and Hickson 2011) Further interest in these structures has been triggered by the discovery that components of the BTR complex (composed of the Bloom syndrome helicase (BLM), TOP3A, and RMI1) co-localize with PICH on UFBs (Chan et al 2007; Hutchins et al 2010). A subpopulation of non-centromere-derived UFBs is characterized by co-localization of PICH with the Fanconi anemia proteins FANCD2 and FANCI (Chan et al 2009a; Naim and Rosselli 2009). These Fanconi anemia proteins associate with fragile site loci and presumably mark abnormally intertwined DNA structures induced by replication stress. The association of PICH with the BTR complex and Fanconi anemia proteins suggests that the processing and resolution of UFBs plays an important role in the maintenance of genome stability
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